This project is designed to assess the physiological importance of calcium ions in the regulation of energy metabolism. We have applied the fluorescent chelating-agent protocol, which was developed for the measurement cytosolic free Ca2+, to the measurement of mitochondrial matrix free Ca ((Ca2+))m. This allowed us to study directly the relationship between (Ca2+)m and the activation of the matrix enzyme pyruvate dehydrogenase for the first time in studies with isolated rat cardiac mitochondria loaded with indo-1. The KO.5 for activation was found to be 300 nM Ca2: this value was unaffected by procedures, e.g. omission of Mg2+, Na+, which change the magnitude of the Ca concentration gradient across the mitochondrial membrane. We have also studied the degree of activation of pyruvate dehydrogenase as a function of the rate of electrical stimulation of isolated guinea pig hearts: in a parallel study, mitochondria were rapidly isolated from stimulated hearts, using a protocol which prevents loss or gain of mitochondrial Ca and allows us to relate this quantity to the level of dehydrogenase activation. As the significance of dehydrogenase activation lies in the NADH/NAD ratio which can be maintained in the face of elevated work -loads, we have also measured the ratio NADH/NAD in suspensions of rat heart mitochondria with saturating and non-saturating concentrations of pyruvate or 2-oxoglutarate as respiratory substrate, in the presence or absence of Ca2+, and correlated this with the flux through oxidative phosphorylation. Finally, we have examined the ability of cardiac mitochondria from senescent dogs to oxidize substrates with emphasis on fatty acids) as a function of lifestyle, viz-sedentary versus exercised.